Treatment for lipodystrophy

ABSTRACT

The present invention provides a therapeutic compound of formula (I) and their pharmaceutically acceptable salts for the prevention and treatment of lipodystrophy caused because of HIV infection or combination therapy of HIV-1 protease inhibitors (Pis) and/or reverse transcriptase inhibitors (nRTIs) by neutralizing lipohypertrophy, lipoatrophy and metabolic abnormalities in HIV patient.

This application is the U.S. national phase of International ApplicationNo. PCT/IN2012/000069 filed 30 Jan. 2012 which designated the U.S. andclaims priority to IN 257/MUM/2011 filed 31 Jan. 2011, the entirecontents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention is related to the development of therapeuticcompound for prevention and treatment of lipodystrophy. In particularthe invention relates to the development of therapeutic compound forprevention and treatment of lipodystrophy in HIV-infected patients(LDHIV). Specifically, the present invention further provides a suitablecomposition useful in the treatment or prevention or alleviation of thesymptoms of lipodystrophy in HIV infected patients (LDHIV)

BACKGROUND OF THE INVENTION

Lipodystrophy is a very dreadful disease and has become a major globalhealth problem. It is a disorder of fat metabolism which causeslipohypertrophy, Lipoatrophy and Metabolic abnormalities. Moreover,lipohypertrophy includes the enlargement of dorsocervical fat pad(commonly called “buffalo hump”), expansion of the circumference of theneck by 5-10 cm, hypertrophy occurring in breast, central truncaladiposity resulting from abdominal visceral fat accumulation, symmetricand asymmetric lipomatoses. A rare pattern of lipoaccumulation involvesformation of band like lipomatosis tissue symmetrically from thebreasts, laterally to the axillae, Suprapubic fat pads (pubic lipomas)and the development of multiple angiolipomas.

Lipoatrophy includes a temporal wasting and loss of subcutaneous fatfrom the cheeks (buccal fat pad) which produces an emaciated appearancewith prominent nasolabial creases. Further subcutaneous tissue isdepleted from the arms, shoulders, thighs, and buttocks (peripheralwasting), with prominence of the superficial veins in these sites.

Metabolic abnormalities include augmentation in cholesterol andtriglyceride levels and reduced high-density lipoprotein (HDL)cholesterol levels, Insulin resistance, type 2 diabetes mellitus, andlactic academia.

Lipodystrophy is very commonly associated with the HIV patients who arebeing treated anti-retroviral medicines. Such medicines can includeHIV-1 protease inhibitors (PIs), Nucleoside reverse transcriptaseinhibitors (NRTIs), Non-nucleoside Reverse Transcriptase Inhibitors(NNRTIs), Fusion Inhibitors, Entry Inhibitors—CCR5 co-receptorantagonist, HIV integrase strand transfer inhibitors etc. Thesemedicines improve the survival of the patient but also producelipohypertrophy, Lipoatrophy and other Metabolic abnormalities.

HIV-1 protease inhibitors (PIs) appear to be the strongest link tolipodystrophy in HIV-infected patients LDHIV as it inhibits maturationof sterol response element binding proteins (SREBP), which affectintracellular fatty acid and glucose metabolism and adipocytedifferentiation (Mallon et al, J Infect Dis, 2005). Furthermore, the PIsalso down-regulate peroxisome proliferator-activated receptor gamma(PPAR γ), an important nuclear transcription factor that is affected bySREBPs and is necessary for adipocyte differentiation and function andfatty acid metabolism.

Other factors, such as duration of HIV infection, age, and gender, mayalso contribute to the risk of development of LDHIV. The molecular basisof LDHIV is still remains unknown and no specific therapy is availablefor LDHIV.

Reverse transcriptase inhibitors (nRTIs) like stavudine, didanosine andzidovudine may cause mitochondrial toxicity by inhibiting mitochondrialDNA polymerase-γ in fat and other tissues and thus interfering withrespiratory chain complexes. The result is impaired fatty acid oxidationand intracellular accumulation of triglycerides and lactate

In addition, lipodystrophy is also observed in acute HIV infection,lending support to a direct viral role as well. Potential host riskfactors include age, sex, and race or ethnicity. Lipodystrophy is morecommon in older patients; fat accumulation is more common in women andlipoatrophy in men; and non-Hispanic black patients appear to be atlower risk of lipoatrophy. A genetic component is indicated by a recentanalysis in AIDS Clinical Trials Group (ACTG), study 5005s, suggestingeither predisposition or protection associated with mitochondrial DNApolymorphisms. Hulgan et al, J Infect Dis, 2008 describes that patientshomozygous for C/C at the HFE187 locus (n=71) had a 0.6-kg and 12.5%loss of limb fat at weeks 48 to 64, with 37 (52%) of the 71 patientsdiagnosed with clinical lipoatrophy. By comparison, heterozygouspatients with HFE187C/G had a 0.2-kg and 6.1% increase in limb fat, with6 (26%) of 23 patients having clinical lipoatrophy (P<0.05 for allcomparisons).

A number of strategies for reducing central obesity have beeninvestigated such as stopping PI treatment but it is not effective.Changes in diet and exercise have produced improvements, but adherenceto a regimen of lifestyle change is difficult for most patients.Liposuction may be applied particularly with dorsocervical fataccumulation, i.e., “buffalo hump”.

It is evident from the several studies that thiazolidinediones show nochange in VAT (Pathogenesis and treatment of lipodystrophy, vol. 16,issue 4, October/November, 2004)

Testosterone replacement to physiologic levels reduces visceral adiposetissue (VAT), total fat, and abdominal fat and improves insulinsensitivity and lipid profile in older, non-HIV-infected men with upperbody obesity and low testosterone levels. In a recent study, 88HIV-infected men with central obesity (waist circumference>100 cm) andlow testosterone levels (<400 ng/dL) underwent randomization totestosterone as a transdermal gel at a dose of 10 g daily or placebo for24 weeks (Bhasin et al, J Clin Endocrinol Metab, 2007). The testosteronegroup had statistically significant reductions in abdominal fat (−1.5%vs +4.3%), abdominal subcutaneous adipose tissue (SAT) (−7.2% vs +8.1%),trunk fat (−9.9% vs +4.6%), and limb fat (−10.1% vs +3.1%); the latterfinding is of potential concern in a population predisposed tolipoatrophy. No statistically significant difference in change in VAT(+0.9% vs +2.3%) was observed, and no statistically significantdifferences were observed in changes in lipid levels, fasting bloodglucose levels, insulin levels, or insulin resistance.

Like testosterone, growth hormone (GH) has fat-oxidizing and lipolyticproperties. A substantial proportion of HIV patients with centralobesity (approximately 30%-40%) have impaired GH biology, includingreduced GH mass secretion, reduced response to GH releasing hormone(GHRH) and free fatty acids, and increased somatostatin tone, whichsuppresses GH. A number of recent studies have assessed GH treatment inHIV patients with fat accumulation. In 1 study, 325 HIV patients withincreased waist: hip ratios and increased VAT measurements received.

Although, the growth hormone (GH) and GH releasing hormone (GHRH)therapies show some promising result as they have fat-oxidizing andlipolytic properties however, there are limitations to their use. Theyare parenteral therapies and either expensive (rhGH) or not FDA-approved(tesamorelin). Thus far, there is evidence of waning durability of thereduction in VAT after their discontinuation, short-term increases ininsulin resistance with rhGH, and small short-term reductions.

Recent research publications have shown the use of two lipid-loweringclasses of drugs, statins and fibrates, antiretroviral switchingstrategies and use of insulin-sensitising drugs as having somebeneficial effect on lipodystrophy. However, no single therapy is ableto reach desirable clinical end point for HIV associated lipodystrophy.

Hence it is desirable to develop a compound which can overcome the abovediscussed drawback associated with prior art and develop a therapy forHIV associated lipodystrophy.

Hypolipidemic agents which are PPAR modulators have been disclosed in WO91/19702, WO 94/01420, WO 94/13650. WO 95/03038, WO 95/17394, WO96/04260, WO 96/04261, WO 96/33998, WO 97/25042, WO 97/36579, WO98/28534, WO 99/08501, WO 99/16758, WO 99/19313, WO99/20614, WO00/23417, WO 00/23445, WO 00/23451, WO 01/53257.

WO 03009841 discloses compounds of the following general formula

These compounds are reported to be hypolipidaemic agents. This documentalso discloses sodium and calcium salts of some of the compoundsdisclosed therein. However, the sodium salts of the compounds of thepresent invention was difficult to isolate due to rapid degradationwhile the Calcium salt was poorly absorbed limiting its efficacy andpossibility of further development. Further, the calcium salt was alsofound to degrade on long term storage. It has surprisingly now beenfound that certain compounds and their selected salts are effective inthe treatment of lipohypertrophy, lipoatrophy and metabolicabnormalities in HIV patients.

Embodiments of the Invention

In an embodiment the present invention provides a compound of formula(I) suitable for the treatment and prevention of lipodystrophy.

In an embodiment, the conditions associated with lipodystrophy includesthe symptoms of lipohypertrophy, lipoatrophy and other metabolicabnormalities.

In another embodiment, the present invention provides a compound offormula (I) for the treatment and prevention or alleviation of symptomsof lipohypertrophy, lipoatrophy and metabolic abnormalities in HIVpatient.

In yet another embodiment the present invention provides theadministration of compound of formula (I) and their pharmaceuticallyacceptable salts alone or in combination with other suitable agents astherapeutic agent for the treatment and prevention alleviation ofsymptoms of lipodystrophy.

In yet another embodiment the present invention provides a suitablecomposition comprising the compound of formula (I) or their suitablepharmaceutical compositions suitable for the treatment and preventionalleviation of symptoms of lipodystrophy.

In another embodiment, the present invention provides for certainpharmaceutical salts of compound of formula (I).

SUMMARY OF THE INVENTION

The present invention provides a compound of formula (I) and theirpharmaceutically acceptable salts for the prevention and treatment oralleviation of symptoms of lipodystrophy. The present invention providesa compound of formula (I) and their pharmaceutically acceptable saltsfor the prevention and treatment or alleviation of symptoms oflipodystrophy caused either because of HIV infection or due to treatmentwith anti-retrovirals. Such anti-retrovirals can include HIV-1 proteaseinhibitors (PIs), Nucleoside reverse transcriptase inhibitors (NRTIs),Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs), FusionInhibitors, Entry Inhibitors—CCR5 co-receptor antagonist, HIV integrasestrand transfer inhibitors etc. or combination therapy involving one ormore anti-retrovirals. The compound of formula (I) neutralizeslipohypertrophy, lipoatrophy and metabolic abnormalities in HIV patient.Moreover, the present invention also provides a suitable compositioncomprising compound of formula (I) useful in the treatment or preventionor alleviation of the symptoms of lipodystrophy in HIV infected patients(LDHIV).

In a further embodiment are disclosed certain new salts corresponding tothe compound of formula (I) wherein M represents K or Mg.

DESCRIPTION OF THE INVENTION

The present invention describes compound of formula (I) which issuitable for the treatment of lipodystrophy or HIV associatedlipodystrophy.

wherein ‘R’ is selected from hydroxy, hydroxyalkyl, acyl, alkoxy,alkylthio, thioalkyl, aryloxy, arylthio and M⁺ represents suitable metalcations such as Na⁺, K⁺, Ca⁺², Mg⁺², and the like.

In a preferred embodiment, ‘R’ represents thioalkyl, alkoxy orhydroxyalkyl group; In a still preferred embodiment. ‘R’ represents—SCH₃ or —OCH₃ group.

In an embodiment is provided suitable pharmaceutical composition for thetreatment of lipodystrophy or HIV associated lipodystrophy comprisingthe compound of formula (I). The pharmaceutical composition of thepresent invention comprises compound of formula (I) along with suitableexcipients as defined hereinafter for the treatment of lipodystrophy orHIV associated lipodystrophy.

In another embodiment, the present invention provides a method oftreating a subject suffering from lipodystrophy or HIV associatedlipodystrophy which comprises treatment of a patient in need of suchtherapy, with compound of formula (I) or suitable pharmaceuticalcompositions containing them.

In a further embodiment the present invention provides use of thecompound of formula (I) or their suitable pharmaceutical compositionsfor the treatment of lipodystrophy or HIV associated lipodystrophy.

In an embodiment the present invention provides certain new salts ofcompound of formula (Ia)

wherein ‘R’ is selected from hydroxy, hydroxyalkyl, acyl, alkoxy,alkylthio, thioalkyl, aryloxy, arylthio and M⁺ represents suitable metalcations selected from K⁺, Mg⁺².

In a preferred embodiment, ‘R’ represents thioalkyl and alkoxy orhydroxyalkyl group; In a still preferred embodiment, ‘R’ represents—SCH₃ or —OCH₃ group.

In another preferred embodiment, M⁺ represents Mg⁺².

The effective amount of the said compound of formula (I) is selectedfrom 1 mg to 500 mg preferably 1 mg to 250 mg and more preferably 4 mgto 50 mg. The compound of formula (I) or its suitable salts isadministrated orally, intravenously, parentally in the subject who is inneed of treatment.

In an embodiment the compound of formula (I) is useful for the treatmentor prevention or alleviation of the symptoms of lipodystrophy. In apreferred embodiment the compound of formula (I) is useful in thetreatment or prevention or alleviation of the symptoms of HIV associatedlipodystrophy. In such embodiment the Lipodystrophy is a disorder of fatmetabolism which causes lipohypertrophy lipoatrophy and metabolicabnormalities.

In an embodiment the compound of formula (1) cure or prevent oralleviate at least one symptoms of lipodystrophy including, but notlimited to, acting as an agent for lowering &/or control blood glucoselevels, an agent used to control lipid levels, e.g., as an agent used tolower control cholesterol, an antioxidant, an appetite suppressingagent, an anti-obesity agent, a probiotic or an anti-inflammatory agent.In another embodiment the compound of formula (1) cure or prevent oralleviate at least one symptoms of lipodystrophy including, but notlimited to triglyceride level, VLDL level and Apo B level in serum. Inanother embodiment the compound of formula (1) cure or prevent oflipodystrophy by improving at least one of the condition selected fromHDL level, Apo A1 level, HOMA of beta cell function derived fromc-peptide.

In an embodiment the present invention also provides a suitablepharmaceutical composition of compounds of formula (I) or theirderivative. The pharmaceutical composition of the present inventionessentially comprises of

-   -   the pharmaceutically active substance;    -   a suitable buffering agent;    -   a suitable stabilizer;    -   optionally with one or more pharmaceutically acceptable        excipients.

The suitable stabilizers used in pharmaceutical composition are selectedfrom Polacrilin potassium, Potassium chloride, Sodium stearyl fumarateand preferably selected from Sodium stearyl fumarate. The suitablebuffering agent are selected from sodium acetate, ammonia solution,ammonium carbonate, sodium borate, adipic Acid, glycine, monosodiumglutamate and preferably selected from ammonia solution.

The pharmaceutically acceptable excipients are selected at least onefrom carriers, binders, antioxidant agents, disintegrating agents,wetting agents, lubricating agents, chelating agents, surface activeagents, and the like.

Diluents include, but are not limited to lactose monohydrate, lactose,polymethacrylates selected from Eudragit, potassium chloride,sulfobutylether b-cyclodextrin, sodium chloride, spray dried lactose,and preferably sulfobutyl ether b-cyclodextrin. Carriers include, butare not limited to lactose, white sugar, sodium chloride, glucose, urea,starch, calcium carbonate and kaolin, crystalline cellulose, and silicicacid. Binders include, but are not limited to carbomers selected fromcarbopol, gellan, gum Arabic, hydrogenated vegetable oil,polymethacrylates selected from Eudragit, xanthan, lactose and Zein.Antioxidant agents include, but are not limited to, Hypophosphorousacid, Sodium formaldehyde, sodium formaldehyde sulfoxylate, sulfurdioxide, tartaric acid, thymol and methionine. Disintegrating agentsinclude, but are not limited to, bicarbonate salt, chitin, gellan gum,polacrillin potassium and Docusate Sodium. Wetting agents include, butare not limited to, Glycerin, lactose, Docusate Sodium and Glycine,Lubricating agents used include, but are not limited to, Glycerinbehenate, hydrogenated vegetable oil, sodium stearyl fumarate andMyristic Acid. Chelating agents include, but are not limited to, Maltoland Pentetic Acid. Surface active agents include but are not limited to,Nonionic surfactant selected from alkyl polyglucosides, cocamide DEA,cocamide MBA, cocamide TEA, decyl maltoside and octyl glucoside; Anionicsurfactant selected from arachidic acid and arachidonic acid; Cationicsurfactant selected from cetyl trimethylammonium bromide andcetylpyridinium chloride.

In an embodiment the formulation is useful for the treatment orprevention or alleviation of the symptoms of lipodystrophy. In apreferred embodiment the said formulation is useful in the treatment orprevention or alleviation of the symptoms of HIV associatedlipodystrophy.

Lipodystrophy is a disorder of fat metabolism which causeslipohypertrophy, lipoatrophy and metabolic abnormalities. Moreover,lipohypertrophy includes the enlargement of dorsocervical fat pad(commonly called “buffalo hump”), expansion of the circumference of theneck by 5-10 cm, hypertrophy occurs in breast, Central truncal adiposityresults from abdominal visceral fat accumulation, symmetric andasymmetric lipomatoses. A rare pattern of lipoaccumulation involvingbandlike lipomatosis tissue symmetrically from the breasts, laterally tothe axillae, suprapubic fat pads (pubic lipomas) and the development ofmultiple angiolipomas.

Lipoatrophy includes a temporal wasting and loss of subcutaneous fatfrom the cheeks (buccal fat pad) produces an emaciated appearance withprominent nasolabial creases, subcutaneous tissue is depleted from thearms, shoulders, thighs, and buttocks (peripheral wasting), withprominence of the superficial veins in these sites.

Metabolic abnormalities include augmentation in cholesterol andtriglyceride levels and reduced high-density lipoprotein (HDL)cholesterol levels. Insulin resistance, type 2 diabetes mellitus, andlactic academia.

The compounds of the present invention due to their beneficial effect onlipodystrophy, will have beneficial effect on Body fat redistribution(Lioatrophy or Hypertrophy or abnormal distribution), Dyslipidemia,Glucose homeostatis, Pro-inflammatory conditions, impact on morbidityand mortality, impact on quality of life, impact on patient's reportedoutcomes like self perception etc

Moreover, the precise mechanisms underlying this syndrome are not wellunderstood, several hypotheses based on in vitro and human studies mayexplain the pathogenesis of the changes. Some experts presently believethat HIV type 1 (HIV-1) protease inhibitors (PIs) and nucleoside reversetranscriptase inhibitors (NRTIs), especially stavudine and zidovudine,are implicated as follows:

-   (i) decreased production of retinoic acid and triglyceride uptake:    PIs have a high affinity for the catalytic site of HIV-1 protease,    which shares a 60% sequence homology with 2 proteins involved in    lipid metabolism, cytoplasmic retinoic acid-binding protein type 1    (CRABP-1) and low-density lipoprotein receptor-related protein    (LDLR-RP). Inhibition of CRABP-1 impairs the production of retinoic    acid, leading to decreased fat storage and adipocyte apoptosis with    the subsequent release of lipids into the circulation. Inhibition of    LDLR-RP results in hyperlipidemia secondary to the failure of    hepatic and endothelial removal of chylomicrons and triglycerides    from the circulation.-   (ii) inhibition of mitochondrial DNA (mtDNA) polymerase gamma: NRTIs    inhibit mtDNA polymerase gamma, leading to mtDNA depletion,    respiratory chain dysfunction, and reduced energy production, which,    in turn, causes insulin resistance and secondary dyslipidemia.    Interestingly, mtDNA is depleted only at normal oxygen    levels—hypoxic adipocytes do not take up triglycerides and are    resistant to mtDNA-induced damage, except after treatment with    NRTIs.-   (iii) inhibition of lipid metabolism: Some PIs, particularly    ritonavir, inhibit cytochrome P450 3A, a key enzyme in lipid    metabolism.-   (iv) prevention of the development of adipocytes: Saquinavir,    ritonavir, and nelfinavir (all PIs) directly inhibit the development    of adipocytes from stem cells and increase the metabolic destruction    of fat in existing adipocytes.

In an embodiment the compound of formula (I) or pharmaceuticalcomposition containing the compound of formula (I) cure or prevent oralleviate at least one symptoms of lipodystrophy including, but notlimited to, acting as an agent for lowering &/or an agent used tocontrol blood glucose levels, an agent used to control lipid levels,e.g., as an agent used to lower control cholesterol, an antioxidant, anappetite suppressing agent, an anti-obesity agent, anantibiotic/probiotic or an anti-inflammatory agent. In anotherembodiment the pharmaceutical composition cure or prevent or alleviateat least one symptoms of lipodystrophy including, but not limited totriglyceride level, VLDL level and Apo B level in serum. In anotherembodiment the pharmaceutical composition cure or prevent oflipodystrophy by improving at least one of the condition selected fromHDL level, Apo A1 level, HOMA of beta cell function derived fromc-peptide.

In another embodiment the compounds according to Formula (I) can be usedalone or in combination e.g., as an adjunct therapy, with at least oneother therapeutic agent. Compound according to formula (I) can beco-administered with a therapeutic agent used to reduce one or more ofthe symptoms of lipodystrophy including, but not limited to, an agentused to control blood glucose levels, an agent used to control lipidlevels, e.g., an agent used to lower control cholesterol, anantioxidant, an appetite suppressing agent, an anti-obesity agent anantibiotic/probiotic or an anti-inflammatory agent. Such combinationtreatment may be adjunct to anti-retroviral therapy. In a preferredembodiment the compound of formula (I) administrated alone or incombination for the treatment of lipohypertrophy, lipoatrophy andMetabolic abnormalities in HIV patient.

The compound of the present invention when M+ represents K, Mg can beprepared by the processes disclosed herein below along with suitablemodifications known to a skilled person.

Example 1 Preparation of(S)-α-Ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzene-propanoicacid ethyl ester

In a dry, 5 L round bottom flask 2.1 L toluene was taken under nitrogen.To this 366.1 g ethyl (S)-α-2-ethoxy-3-(4-hydroxyphenyl)propionate wasadded at room temperature.

The reaction mixture was stirred under heating, using Dean-starkapparatus, to remove water azeotropically. The reaction mixture wascooled to 50° C. To this was added 319 g anhydrous potassium carbonateand stirred at 90-92° C. for 1 hr. Cooled to 65° C. and added 500 g2-(2-methyl-5-(4-(methylthio)phenyl)-1H-pyrrol-1-yl)ethylmethanesulfonate and 22 g tetra butyl ammonium bromide. Reaction mixturewas heated to 87-92° C. and stirred for 46 hrs. Cooled to 70-75° C.,added 1.5 L toluene, charcoalised using 75 g charcoal and cooled to roomtemperature. Filtrate washed with alkaline solution, washed with water,dried over sodium sulfate and concentrated under vacuum to obtain(S)-α-Ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzene-propanoicacid ethyl ester.

Yield: 650 g, HPLC purity: 84.10%; % Yield 76.0%.

Example 2 Preparation of(S)-α-Ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzenepropanoicacid magnesium salt

In a dry, 250 mL round bottom flask 80 mL methanol was taken. To this 20g(S)-α-ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzene-propanoicacid ethyl ester was added at room temperature, under nitrogen. To this1.89 g sodium hydroxide dissolved in 20 mL water was added and stirredat room temperature for 3 hours to complete hydrolysis. Solvent wasremoved under reduced pressure. 150 mL water was added to concentratethe material. Impurity was removed by solvent washing. To aqueous layerwas added 5 g magnesium acetate tetrahydrate (dissolved in 20 mL water)and stirred with for 15 min. Sticky material was extracted withdichloromethane and subsequently add n-heptane to precipitate(S)-α-ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzenepropanoicacid magnesium salt. Solid was filtered, and dried.

Yield: 10.3 g; HPLC Purity: 98.32%; Chiral purity: 97.64%.

Following the process similar to those described in Examples 1 & 2 thefollowing batchesof)-α-Ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzenepropanoicacid magnesium salt were prepared.

HPLC Chiral No. Batch no. Input Output % Yield purity purity 1 Example 310 g 5.02 g 61.21% 98.22% 98.58% 2 Example 4 10 g 4.97 g 60.68% 97.91% —3 Example 5 15 g 7.34 g 61.94% 98.20% — 4 Example 6 15 g 8.38 g 67.50%99.05% Similar reaction carried out using Magnesium chloride 5 Example 710 g  6.5 g 79.25% 98.53% 99.32% Similar reaction carried out usingMagnesium sulfate 6 Example 8 10 g  6.8 g 82.91%  98.5%

The present invention further discloses use of said compound of formula(I) or their suitable pharmaceutical compositions for the treatment oflipohypertrophy, lipoatrophy and metabolic abnormalities in HIV patient.

Example 9(S)-α-Ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzenepropanoicAcid Potassium Salt

In a dry, 250 mL round bottom flask 72 mL ethyl acetate was taken. Tothis 10 g (S)-(−)α-1-phenylethylamine salt of(S)-α-ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzene-propanoic acid was added at roomtemperature and subsequently 50 mL water and 4.8 mL dilute hydrochloricacid (water 1:1:35% HCl) was added and stirred at room temperature tillsolid was dissolved. Layer was separated and organic layer was washedwith water, dried over sodium sulfate and solvent removed. 9.2 g oilymass obtained. To this was added 50 mL methanol and stirred undernitrogen. To this was added 1.81 g potassium t-butoxide and was stirredat room temperature for 15 min. Solvent removed and added n-Hexane.Again n-hexane was removed and added methanol. Solvent removed undervacuum. Hygroscopic material obtained. Dried it under vacuum to get(S)-α-ethoxy-4-[2-[-methyl-5-[4-(methylthio)phenyl]-1H-pyrrol-1-yl]ethoxy]benzenepropanoicacid potassium salt.

Yield—7.6 g, (92.77%), HPLC Purity 98.60%, Chiral purity 99.56%

Example 10

Title of Study: A Prospective, Multi-Centric, Open-Label, Single ArmStudy to Evaluate the Safety and Efficacy of 4 mg of compound of formula(I) in Hypertriglyceridemia in HIV Associated Lipodystrophy.

Objectives: The objective of this study was to evaluate the safety andefficacy of 4 mg of compound of formula (I) in hypertriglyceridemia inHIV associated lipodystrophy.

Methodology: This was a prospective, multi-centric, open-label, singlearm study to evaluate the safety and efficacy of 4 mg of compound offormula (I) in hypertriglyceridemia in HIV associated lipodystrophy.

After obtaining informed written consent, subjects withhypertriglyceridemia in HIV associated lipodystrophy, on treatment withHAART for at least 18 months and satisfying the inclusion and exclusioncriteria were enrolled in the study. The subjects received 4 mg ofcompound of formula (I) tablet orally, once daily for a period of 12weeks. During this 12-week program, safety parameters were assessed atweeks 2, 6, and 12 and the efficacy was evaluated at week 6 and 12.Number of patients: Planned: 50, Analyzed: 50Test product: Compound of formula (I)Dose 4 mgDuration of treatment: 12 weeksMode of administration: OralBatch number: EMK328Criteria for evaluation: Efficacy:

The primary efficacy endpoint was to assess the percent change in TGlevels from baseline to Week 6 and Week 12. The secondary efficacyendpoint was the assessment of LDL, VLDL, HDL, Non HDL cholesterol,Total cholesterol, Apo A1, Apo B, and C-peptide and fasting insulin forHOMA beta and HOMA IR.

Safety:

Clinical examination and recording of adverse events (AEs) was done onall visits. Electrocardiogram was recorded at screening visit and atWeek 12. Urine pregnancy test was conducted at screening visit

Haematological examination included haemoglobin, haematocrit, red bloodcell (RBC) count, white blood cell (WBC) count with differential(neutrophils, lymphocytes, monocytes, eosinophils and basophils) andplatelet count.

Biochemistry tests included AST, ALT, ALP, total bilirubin, serumproteins, total albumin and globulin, γ-GTT, BUN, Serum creatinine,serum uric acid, CPK, and urine R/Ms (including microalbuminuria andketonuria).

All laboratory parameters were evaluated at enrolment visit (Week 0) andat Weeks 2, 6, and 12.

Statistical Methods

For the efficacy endpoints, treatment effect was evaluated using ananalysis of variance (ANOVA) model with factors for baseline andtreatment. Treatment effects were estimated using the least-square means(LSM) and 95% confidence intervals (CIs) from the ANOVA model.Statistical significance was defined as a two-sided p-value<0.05. Allother secondary endpoints were analyzed using appropriate statisticalmethods.

For safety analysis the frequency tabulations of abnormal physicalexamination and abnormal clinical laboratory parameters were presentedfor each visit. Summary statistics for clinical laboratory parametersand vital signs were presented for each visit. A list of concomitantmedications taken during the study period was summarised.

Adverse events were coded using the Medical Dictionary for RegulatoryActivities (MedDRA) (Version 14). Adverse events and SAEs weresummarized overall, by system organ class (SOC) and by MedDRA preferredterm for treatment emergent adverse events (TEAEs). All AEs, includingthose arising before or after treatment was included in the listings.Separate listings were provided for SAEs and AEs leading todiscontinuation from the study.

Study Design

This was a safety and efficacy study to evaluate 4 mg of compound offormula (I) in hypertriglyceridemia in HIV associated lipodystrophy.This was exploratory proof of concept study designed to assess the proofof safety and efficacy in intended population. The results of compoundof formula (I) from phase II studies in Dyslipidemia subjectsdemonstrated that compound of formula (I) 4 mg is well tolerated andeffective at once daily dosing. Phase I study demonstrated foodsignificantly affects absorption of compound of formula (I), so drug wasrecommended to be consumed preferably in fasting condition. Based uponthese observations 4 mg once daily in fasted condition was selected forpresent study

Selection of Study Population

Inclusion Criteria

Subjects who satisfied all of the following criteria were eligible forenrolment in the study:

-   -   1. Males and females aged 18-65 years.    -   2. Confirmed diagnosis of HIV1 and on HAART for at least 18        months.    -   3. On stable ART regimen for at least 8 weeks prior to inclusion        in the study and ART regimen not expected to change in next 3        months.    -   4. Subjects clinically diagnosed as HIV lipodystrophy (at least        1 moderate or severe lipodystrophy feature identified by doctor        and patient, except isolated abdominal obesity)    -   5. Triglycerides>200 to 500 mg %.    -   6. CD4 count of >50/mm³    -   7. Subject who had given informed consent for participation in        this trial.        Treatments        Treatments Administered

The study had a single arm. Subjects received 4 mg of compound offormula (I) orally once daily in the morning before breakfast, for aperiod of 12 weeks.

Identity of Investigational Product(s)

Compound of formula (I) is divalent magnesium salt of carboxylic acid inthe form of white, amorphous powder, which is freely soluble in dimethylsulfoxide, dichloromethane, slightly soluble in methanol and insolublein water. The drug was supplied as uncoated tablets of 4 mg of theactive ingredient.

Supply from batch no EMK328 was used during the study. The study drugwas manufactured and packaged in cGMP facility.

Primary Efficacy Variable(s)

The primary efficacy endpoint was to determine the percent change in TGlevels from baseline to Week 6 and Week 12.

Secondary Efficacy Variables

The secondary efficacy endpoint was to determine the percent change inLDL, VLDL, HDL, total cholesterol, non-HDL Cholesterol (measured value),Apo A1, and Apo B, C-peptide and fasting insulin for HOMA beta and HOMAIR levels from baseline to Week 6 and Week 12.

Statistical Methods Planned in the Protocol and Determination of SampleSize

Statistical and Analytical Plans

The demographic and baseline characteristics were summarized forcompound of formula (I) 4 mg treatment arm. For continuous measurementssuch as age, the mean, median, standard deviation (SD) and range weretabulated. For categorical measurements such as gender, the frequencieswere computed.

Efficacy Analyses:

The primary efficacy variable was the reduction in TG at Week 6 and Week12 of the treatment period compared with baseline. The change frombaseline was determined as the difference between the means for thetreatment period (Weeks 6/Weeks 12) and the baseline.

For the efficacy endpoints, treatment effect was evaluated using ananalysis of variance (ANOVA) model with factors for baseline andtreatment. Treatment effects were estimated using the least-square means(LSM) and 95% confidence intervals (CIs) from the ANOVA model.Statistical significance was defined as a two-sided p-value<0.05. Allother secondary endpoints were analyzed using appropriate statisticalmethods.

Intent-to-treat (ITT) and/or Per Protocol (PP) analysis were carried outfor the study. The PP analysis was considered definitive while the ITTanalysis was considered supportive during the trial analysis.

Efficacy Results and Tabulations of Individual Patient Data

Analysis of Efficacy

One subject identified as EHT004 in the study: a 35-year-old male, wasreported with abnormally low levels of HDL (3.95 mg/L) and LDL (6.25mg/L) at Visit 1. Though this subject completed the study and wasassessable for efficacy, it was decided to exclude this subject from theefficacy analyses. Therefore a total of 49 subjects were analyzed, forefficacy.

Primary Endpoints

The percent change from baseline in serum TG levels at Week 6 and Week12 following compound of formula (I) 4 mg was statistically significant(−40.98±4.89 and −45.11±3.60, respectively [p-value: <0.0001, each])(Table 1).

TABLE 1 Analysis of change in Triglyceride (mg/dL) from baseline byvisit COMPOUND OF Laboratory FORMULA (I) Test (Unit) Visit 4 mg (N = 49)TG (mg/dL) Visit 1 n 49 Mean ± SD  301.68 ± 86.99 Median 275.45 Minimum200.10 Maximum 481.42 Visit 3 n 49 (Week 6) Mean ± SD  172.81 ± 106.30Median 147.68 Minimum 42.61 Maximum 631.08 Change from Visit 1 −128.87 ±14.96 (LS Mean ± SE) p-values <0.0001 % Change from Visit 1 −40.98 ±4.89 (LS Mean ± SE) p-values <0.0001 Visit 4 n 49 (Week 12) Mean ± SD 166.97 ± 89.17 Median 145.91 Minimum 46.88 Maximum 387.69 Change fromVisit 1 −134.71 ± 10.78 (LS Mean ± SE) p-values <0.0001 % Change fromVisit 1 −45.11 ± 3.60 (LS Mean ± SE) p-values <0.0001 Key toabbreviations: LSM = least square means; N = number of subjects in thetreatment group; n = number of subjects having non-missing baseline andpost-baseline values; SD = standard deviation; SE = standard error; TG =triglycerides. Note: p-values < 0.05 indicates significant and fromANOVA modelSecondary EndpointsHDL Cholesterol:

There was an increase in the HDL cholesterol levels followingadministration of compound of formula (I) 4 mg. The percent change frombaseline in HDL cholesterol following compound of formula (I) 4 mg atWeek 6 and Week 12 was statistically significant (29.92±5.73 and34.56±6.13, respectively [p-value: <0.0001 each]) (Table 2).

TABLE 2 Analysis of change in HDL Cholesterol (mg/dL) from baseline byvisit COMPOUND OF Laboratory FORMULA(I) Test (Unit) Visit 4 mg (N = 49)HDL Visit 1 n 49 Cholesterol Mean ± SD 35.27 ± 7.85 (mg/dL) Median 34.52Minimum 22.23 Maximum 49.90 Visit 3 n 49 (Week 6) Mean ± SD  44.44 ±14.04 Median 43.36 Minimum 20.13 Maximum 73.50 Change from Visit 1  9.17± 1.99 (LS Mean ± SE) p-values <0.0001 % Change from Visit 1 29.92 ±5.73 (LS Mean ± SE) p-values <0.0001 Visit 4 n 49 (Week 12) Mean ± SD 46.14 ± 14.84 Median 47.70 Minimum 17.61 Maximum 82.89 Change fromVisit 1 10.87 ± 2.08 (LS Mean ± SE) p-values <0.0001 % Change from Visit1 34.56 ± 6.13 (LS Mean ± SE) p-values <0.0001 Key to abbreviations: LSM= least square means; N = number of subjects in the treatment group; n =number of subjects having non-missing baseline and post-baseline values;SD = standard deviation; SE = standard error; HDL = high densitylipoprotein. Note: p-values < 0.05 indicates significant and from ANOVAmodelC-Peptide HOMA of Insulin Resistance:

There was an increase in insulin resistance after treatment withcompound of formula (I). The percent change in HOMA IR from baselinefollowing administration of compound of formula (I) 4 mg at Week 6 andWeek 12 was statistically significant (27.87±4.22 and 58.29±5.74respectively [p-value: <0.0001 each]) (Table 3).

TABLE 3 Analyses of change in HOMA of insulin resistance for C-Peptidefrom baseline by visit COMPOUND OF Laboratory FORMULA(I) Test (Unit)Visit 4 mg (N = 49) Homa of Insulin Visit 1 n 49 Resistance for Mean ±SD 1.59 ± 0.82 C-Peptide Median 1.40 Minimum 0.50 Maximum 3.80 Visit 3 n49 (Week 6) Mean ± SD 1.86 ± 0.77 Median 1.70 Minimum 0.90 Maximum 3.60Change from Visit 1 0.27 ± 0.05 (LS Mean ± SE) p-values <0.0001 % Changefrom Visit 1 27.87 ± 4.22  (LS Mean ± SE) p-values <0.0001 Visit 4 n 49(Week 12) Mean ± SD 2.15 ± 0.62 Median 2.10 Minimum 1.10 Maximum 3.60Change from Visit 1 0.56 ± 0.05 (LS Mean ± SE) p-values <0.0001 % Changefrom Visit 1 58.29 ± 5.74  (LS Mean ± SE) p-values <0.0001 Key toabbreviations: HOMA: homeostasis model assessment, IR: insulinresistance, LSM = least square means; N = number of subjects in thetreatment group; n = number of subjects having non-missing baseline andpost-baseline values; SD = standard deviation; SE = standard error Note:p-values < 0.05 indicates significant and from ANOVA modelInsulin (Fasting):

There was an increase in insulin resistance after treatment withcompound of formula (I). The percent change in Insulin from baselinefollowing administration of compound of formula (I) 4 mg at Week 6 andWeek 12 was statistically significant (23.71±3.55 and 47.10±4.21respectively [p-value: <0.0001 each]) (Table 4).

TABLE 4 Analyses of change in Insulin (fasting) from baseline by visitCOMPOUND OF Laboratory FORMULA (I) Test (Unit) Visit 4 mg (N = 49)Insulin Visit 1 n 49 (fasting) Mean ± SD  9.21 ± 6.26 μu/mL Median 7.40Minimum 2.65 Maximum 28.06 Visit 3 n 49 (Week 6) Mean ± SD 10.42 ± 5.74Median 8.35 Minimum 2.14 Maximum 26.82 Change from Visit 1  1.21 ± 0.22(LS Mean ± SE) p-values <0.0001 % Change from Visit 1 23.71 ± 3.55 (LSMean ± SE) p-values <0.0001 Visit 4 n 49 (Week 12) Mean ± SD 11.40 ±4.45 Median 10.18 Minimum 5.93 Maximum 24.29 Change from Visit 1  2.20 ±0.21 (LS Mean ± SE) p-values <0.0001 % Change from Visit 1 47.10 ± 4.21(LS Mean ± SE) p-values <0.0001 Key to abbreviations: LSM = least squaremeans; N = number of subjects in the treatment group; n = number ofsubjects having non-missing baseline and post-baseline values; SD =standard deviation; SE = standard error Note: p-values < 0.05 indicatessignificant and from ANOVA modelInsulin HOMA of Beta-Cell Function:

There was an increase in HOMA of Beta-cell function derived from Insulinafter treatment with compound of formula (I). The percent change in theHOMA of Beta-cell function derived from Insulin from baseline at Week 6and Week 12 was statistically significant (52.50±14.94 and 45.64±6.22,respectively [p-value: 0.0010 and <0.0001, respectively])(Table 5).

TABLE 5 Analyses of change in HOMA of Beta Cell Function for Insulinfrom baseline by visit COMPOUND OF Laboratory FORMULA(I) Test (Unit)Visit 4 mg (N = 49) HOMA of Beta Visit 1 n 48 Cell Function Mean ± SD107.82 ± 52.85 for Insulin Median 97.25 Minimum 10.20 Maximum 234.50Visit 3 n 49 (Week 6) Mean ± SD 136.41 ± 76.00 Median 116.50 Minimum34.90 Maximum 348.00 Change from Visit 1 29.55 ± 8.76 (LS Mean ± SE)p-values 0.0015 % Change from Visit 1  52.50 ± 14.94 (LS Mean ± SE)p-values 0.0010 Visit 4 n 49 (Week 12) Mean ± SD 137.56 ± 46.11 Median125.60 Minimum 9.80 Maximum 273.30 Change from Visit 1 30.78 ± 4.25 (LSMean ± SE) p-values <0.0001 % Change from Visit 1 45.64 ± 6.22 (LS Mean± SE) p-values <0.0001 Key to abbreviations: HOMA: homeostasis modelassessment, LSM = least square means; N = number of subjects in thetreatment group; n = number of subjects having non-missing baseline andpost-baseline values; SD = standard deviation; SE = standard error Note:p-values < 0.05 indicates significant and from ANOVA modelEfficacy ConclusionsPrimary Endpoint:

-   -   There was a statistically significant reduction from baseline in        serum TG levels at Week 6 and Week 12 following compound of        formula (I) 4 mg (percent change of −40.98±4.89 and −45.11±3.60,        respectively [p value: <0.0001, each])        Secondary Endpoints:    -   There was no statistically significant change in the non-HDL        cholesterol levels from baseline following administration of        compound of formula (I) 4 mg at Week 6 and Week 12 (p-values:        0.3963 and 0.4646, respectively)    -   There was a statistically significant increase in the HDL        cholesterol levels from baseline following administration of        compound of formula (I) 4 mg at Week 6 and Week 12 (percent        change: 29.92±5.73 and 34.56±6.13, respectively [p-value:        <0.0001 each]).    -   There was a statistically significant increase in the HOMA of        Beta-cell function derived from C-peptide from baseline        following administration of compound of formula (I) 4 mg at Week        6 and Week 12 (68.25±25.58 and 71.67±16.20, respectively        [p-value: 0.0104 and <0.0001, respectively]).    -   There was a statistically significant increase in the HOMA of        insulin resistance derived from insulin from baseline after        treatment with compound of formula (I) at Week 6 and Week 12        (percent change: 29.10±3.94 and 42.65±3.79, respectively        [p-value: <0.0001 each]).

Therefore, the compound of the present invention includingpharmaceutical compositions containing the same was found to be usefulfor the treatment of lipohypertrophy, lipoatrophy and Metabolicabnormalities in HIV patients.

We claim:
 1. A method for treating lipodystrophy in a subject in needthereof, the method comprising administering an effective amount of acompound of formula (I)

to the subject thereby to treat the lipodystrophy.
 2. The method ofclaim 1, wherein the lipodystrophy is HIV associated lipodystrophy. 3.The method of claim 2, wherein the HIV associated lipodystrophy causeslipohypertrophy, lipoatrophy or a metabolic abnormality.
 4. The methodof claim 1 further comprising administering to the subject the compoundof formula (I) in combination with a second therapeutic agent.